Correction to: Structural modeling of a novel TERC variant in a patient with aplastic anemia and short telomeres.

The original version of this article contained a mistake in the affiliation of E. Bellacchio. Correct affiliation is presented here.

A recognizable systemic connective tissue disorder with polyvalvular heart dystrophy and dysmorphism associated with TAB2 mutations.

Deletions encompassing TAK1-binding protein 2 (TAB2) associated with isolated and syndromic congenital heart defects. Rare missense variants are found in patients with a similar phenotype as well as in a single individual with frontometaphyseal dysplasia. We describe a family and an additional sporadic patient with polyvalvular heart disease, generalized joint hypermobility and related musculoskeletal complications, soft, velvety and hyperextensible skin, short limbs, hearing impairment, and facial dysmorphism. In the first family, whole-exome sequencing (WES) disclosed the novel TAB2 c.1398dup (p.Thr467Tyrfs*6) variant that eliminates the C-terminal zinc finger domain essential for activation of TAK1 (TGFß-activated kinase 1)-dependent signaling pathways. The sporadic case carryed a ~2 Mb de novo deletion including 28 genes also comprising TAB2. This study reveal an association between TAB2 mutations and a phenotype resembling Ehlers-Danlos syndrome with severe polyvalvular heart disease and subtle facial dysmorphism. Our findings support the existence of a wider spectrum of clinical phenotypes associated with TAB2 perturbations and emphasize the role of TAK1 signaling network in human development.

Identification of a second HOXA2 nonsense mutation in a family with autosomal dominant non-syndromic microtia and distinctive ear morphology.

Microtia is a congenital defect affecting external ears, which appear smaller and sometimes malformed. Here we describe a five-generation family with isolated bilateral microtia segregating as an autosomal dominant trait. Similar features have been previously observed in an autosomal dominant family with non-syndromic microtia and hearing loss segregating with a HOXA2 nonsense variant. HOXA2 biallelic mutations were also described in an inbreed family with autosomal recessive microtia, hearing impairment and incomplete cleft palate. In our family, sequence analysis detected a heterozygous protein truncating nonsense variant [c.670G>T, p.(Glu224*)] segregating in all affected individuals and absent in public databases. This study confirms the role of HOXA2 gene in dominant isolated microtia and contribute to further define the dysmorphogenetic effect of this gene on ear development.

Clinical and molecular characterization of two patients with palmoplantar keratoderma-congenital alopecia syndrome type 2.

Palmoplantar keratoderma-congenital alopecia (PPKCA) syndrome is a rare genodermatosis, with two clinically recognizable forms: dominant (Type 1) and recessive (Type 2). Reports of only 18 patients have been published to date, and the molecular basis of the condition is unknown. We describe two cases with PPKCA Type 2 (PPKCA2), comprising a novel patient, originally reported as an example of autosomal ichthyosis follicularis-atrichia-photophobia syndrome, and the 6-year follow-up of a previously published case. Extensive molecular studies of both patients excluded mutations in all the known genes associated with PPK and partially overlapping syndromes. The striking similarities between these two patients confirm PPKCA2 as a discrete genodermatosis, of which the main features are congenital and universal alopecia, diffuse keratosis pilaris, facial erythema, and a specific PPK with predominant involvement of the fingertips and borders of the hands and feet, with evolution of sclerodactyly, contractures and constrictions. Clinical follow-up of these patients has demonstrated progressive worsening of the hand involvement and attenuation of facial erythema.

Detection of a novel HLA-B allele, HLA-B*08:111, in an Italian bone marrow donor.

The newly detected HLA-B*08:111 allele shows two nucleotide differences from B*08:01:01 in codons 113 and 114.

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011.

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

Severe Bartter syndrome type 1: Prompt postnatal management thanks to antenatal identification of SLC12A1 pathogenic variants.

Bartter syndrome (BS) refers to a group of hereditary kidney disorders. One antenatal form is Bartter syndrome type 1 (BS1), caused by pathogenic variants in the SLC12A1 gene. We report a case of BS1 presenting with severe polyhydramnios. The fetus was found to carry three pathogenic variants of SLC12A1, leading to the antenatal diagnosis of BS1 and its prompt management. At age 18 days, clinical conditions were complicated by the onset of sepsis requiring supportive measures as well as steroid and antibiotic therapy. Any newborn with an antenatal history of polyhydramnios or postnatal polyuria should be suspected of having BS, since delayed diagnosis may lead to rapid renal failure.

Novel and recurrent p14 mutations in Italian familial melanoma.

CDKN2A and CDK4 are the only known high-penetrant genes conferring proneness to cutaneous melanoma. The CDKN2A locus consists of four exons and encodes several alternate transcripts, two of which are p16(INK4a) and p14(ARF), and originate from different open reading frames. Exon 1alpha is specific for p16(INK4a), while exon 1beta characterizes p14(ARF). Most CDKN2A mutations are located in exons 1alpha and 2, while exon 1beta variations have been identified in rare melanoma-prone pedigrees. In a previous study, we investigated 155 Italian melanoma cases, including 94 familial melanomas (FAMs) and 61 sporadic multiple primary melanomas (MPMs), for p16(INK4a)/CDK4 germline alterations and identified 15 p16(INK4a) and 1 CDK4 point mutations. In the present work, we extended our search to p14(ARF) mutations and CDKN2A deletions in the remaining samples. We identified the recurrent g.193+1G> A mutation in two FAM cases, while an additional pedigree displayed the previously undescribed variant g.161G> A. Multiplex ligation-dependent probe amplification (MLPA) screening for copy variations resulted negative in all cases. In Italy, the overall frequency of p14(ARF) mutations is 3.2% in FAM and 0% in sporadic MPM. Re-evaluation of our patients' cohort emphasizes that the chance of identifying CDKN2A/CDK4 mutations in FAM is mainly influenced by the number of affected family members and the presence of one or more MPM cases. Accordingly, mutation rate rises to 61% in selected cases. Further studies are expected in order to investigate CDKN2A rarer mutations, including atypical deletions and inherited epimutations.

Complex multipathways alterations and oxidative stress are associated with Hailey-Hailey disease.

BACKGROUND: Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesions. While a strong relationship exists between mutations in the gene that encodes the Ca(2+)/Mn(2+)-adenosine triphosphatase ATP2C1 and HHD, we still have little understanding of how these mutations affect manifestations of the disease. OBJECTIVES: This study was designed to determine early signalling events that affect epithelial cell growth and differentiation during HHD development. METHODS: Expression of key regulatory signals important for maintaining skin homeostasis were evaluated by Western blot analysis and by reverse transcriptase-polymerase chain reaction in primary keratinocytes obtained from skin biopsies of patients with HHD. Reactive oxygen species accumulation in primary keratinocytes derived from lesional skin of patients with HHD was assessed by dihydrorhodamine 123 (DHR) assay. RESULTS: HHD-derived keratinocytes showed downregulation of both Notch1 and differential regulation of different p63 isoforms. Itch and p63 are co-expressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. We found that the Itch protein was significantly decreased in HHD-derived keratinocytes whereas the expression of its target, c-Jun, remained unaffected. We also found that HHD-derived keratinocytes undergo oxidative stress, which may explain both Notch1 and Itch downregulation. CONCLUSIONS: Our attempt to explore the molecular mechanism underlying HHD indicates a complex puzzle in which multi-hit combinations of altered signal pathways may explain the wide spectrum of defects in HHD.

Molecular characterization of 6 unrelated Italian patients with 5alpha-reductase type 2 deficiency.

Steroid 5alpha-reductase (5alphaR) deficiency (OMIM number #264600) is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alphaR type 2 gene (SRD5A2) resulting in dihydrotestosterone deficiency during fetal development. We report on the analysis of the SRD5A2 gene in 6 unrelated 46,XY Italian patients with external genitalia morphology ranging from predominantly female to nearly completely male. Three subjects were seen and assessed at birth, 1 patient was referred to us before puberty, and 2 at postpubertal age. Six different causative mutations (5 missense and 1 nonsense) and a rare polymorphism were identified. Four patients presented homozygous single-base substitutions. These SRD5A2 mutations were located in exon 2 (variant Cys133Gly), exon 4 (Gly196Ser and Ala207Asp) and exon 5 (Tyr235Phe). A fifth subject was a compound heterozygote who carried a nonsense mutation in exon 1 (Trp53X) and a second SRD5A2 alteration in exon 5 (Tyr235Phe). The final patient presented a mutation in only 1 allele (Gly34Trp) together with the Ala49Thr variant. The molecular characterization of these patients made it possible to identify novel mutations and to confirm, before gender assignment or any surgical approach, the suspected 5alphaR deficiency in 2 newborns, 1 of whom had inconclusive hormonal data. 5alphaR deficiency in subjects without parental consanguinity and the presence of compound heterozygotic patients suggest that SRD5A2 mutations carrier frequency may be higher than previously thought.

A novel Leu153Ser mutation of the Fanconi anemia FANCD2 gene is associated with severe chemotherapy toxicity in a pediatric T-cell acute lymphoblastic leukemia.

Fanconi anemia (FA) is an autosomal recessive disease characterized by pancitopenia, congenital malformations, predisposition to cancers and chromosomal instability. We report the clinical and molecular features of a patient initially identified as a potential FA case only because of chemotherapy toxicity during the treatment of a T-lineage acute lymphoblastic leukemia (ALL). Cells from this patient showed a moderate chromosomal instability, increasing sensitivity to DNA crosslinking agents but normal response to ionizing radiation. The analysis of FA proteins demonstrated a marked reduction of FANCD2 (>95%), but normal levels of FANCA or FANCG. Interestingly, this defect was associated with a homozygous missense mutation of FANCD2, resulting in a novel amino-acid substitution (Leu153Ser) at residue Leu153, which is highly conserved through evolution. The FANCD2(L153S) protein, whose reduced expression was not due to impaired transcription, was detected also in its monoubiquitinated form in the nucleus, suggesting that the mutation does not affect post-translation modifications or subcellular localization but rather the stability of FANCD2. Therefore, the hypomorphic Leu153Ser mutation represents the first example of a FANCD2 defect that might promote clonal progression of tumors, such as T-ALL, and severe chemotherapy toxicity in patients without any clinical manifestations typical of FA.

Homozygous p.M172K mutation of the TFR2 gene in an Italian family with type 3 hereditary hemochromatosis and early onset iron overload.

The p.M172K TFR2 mutation was identified in two Italian siblings aged 32 and 40 years old with primary iron overload. The two patients showed a severe increase in serum iron indices. From the age of 25, the male sib also revealed abnormal levels of hepatic enzymes, presumably in relation to iron induced liver damage. Clinical findings seem to evidence that type 3 hemochromatosis can be more serious than classic hemochromatosis. This report adds two more type 3 hereditary hemochromatosis cases which suggest that TFR2 mutations could be more frequently involved in non-HFE hemochromatosis than has been actually thought.

HAMP gene mutation c.208T>C (p.C70R) identified in an Italian patient with severe hereditary hemochromatosis.

Hepcidin is a recently identified hormone peptide involved in regulation of iron homeostasis. HAMP gene mutations have been described to date in five families with iron overload. We have identified the c.208T>C (p.C70R) mutation in the HAMP gene in a patient affected by a severe form of hereditary hemochromatosis. The variant, occurring in a highly conserved amino acid, disrupts one of the 4 intramolecular disulphide bonds present in hepcidin molecules of all vertebrates, and is presumably able to destabilize the peptide structure. The investigated patient was also found to harbor a heterozygous HFE c.845G>A (p.C282Y) mutation that may have contributed in increasing his iron burden.

Two novel mutations and a new STK11/LKB1 gene isoform in Peutz-Jeghers patients.

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disorder with variable expression and incomplete penetrance characterized by mucocutaneous pigmentation, predisposition to hamartomatous intestinal polyposis, and various other neoplasms. It occurs in approximately 1 in 8,300 to 29,000 live births. In nearly 50% of patients PJS is caused by germ line mutations in the STK11/LKB1 serine/threonine kinase gene, the only kinase gene currently known to act as a tumor suppressor. We have performed a mutation search in the STK11/LKB1 gene in 8 sporadic cases and 3 PJS families using a combination of different screening techniques. We have identified four mutations, two of which I177N and the IVS2+1A->G, were previously unreported. We have also evaluated the presence of cDNA alterations by means of RT-PCR analysis and direct cDNA sequencing and have found two aberrant transcripts in a single PJS case despite the lack of any apparent genomic alteration. Finally, we report the presence of a novel STK11/LKB1 cDNA isoform observed in all the normal subjects studied as well as in the majority of the PJS patients.

Imbalance in the antioxidant pool in melanoma cells and normal melanocytes from patients with melanoma.

In order to evaluate the free radical defense systems of melanocytes and their possible correlation with melanoma, we have studied in cultured normal human melanocytes (20), normal melanocytes from melanoma patients (15), and melanoma cells (40) the fatty acid pattern of membrane phospholipids as a target of peroxidative damage and the superoxide dismutase and catalase activities, vitamin E, and ubiquinone levels as intracellular antioxidants. Cells were cultured in the same medium and analyzed at III or IV passage. Compared to the values obtained in normal human melanocytes, melanoma cells showed on average: a) higher levels of polyunsaturated fatty acids, b) increased superoxide dismutase and decreased catalase activities, higher vitamin E, and lower ubiquinone levels. Among the normal melanocytes from melanoma patients studied, two groups were differentiated: a) cultures (7) with enzymatic and non-enzymatic antioxidants level similar to those of normal human melanocytes; b) cultures (8) with antioxidant patterns similar to those observed in melanoma cells. Polyunsaturated fatty acids were also increased in the latter group. The results indicate that in melanoma cells and in a percentage of normal melanocytes from melanoma patients, an imbalance in the antioxidant system can be detected that can lead to endogenous generation of reactive oxygen species and to cellular incapability of coping with exogenous peroxidative attacks. These alterations could be correlated with the malignant transformation of cells and with the progression of the disease.

Increased sensitivity to peroxidative agents as a possible pathogenic factor of melanocyte damage in vitiligo.

To examine the sensitivity of vitiligo melanocytes to external oxidative stress, we studied enzymatic and non-enzymatic anti-oxidants in cultured melanocytes of normal subjects (n = 20) and melanocytes from apparently normal skin of vitiligo patients (n = 10). The activity of superoxide dismutase and catalase and the intracellular concentrations of vitamin E and ubiquinone were evaluated in cultures at the fourth or fifth passage. In addition, cells were exposed to various concentrations of a peroxidizing agent, cumene hydroperoxide (CUH, 0.66-20 microM), for 1 and 24 h. Compared to normal melanocytes, vitiligo melanocytes showed normal superoxide dismutase and significantly lower catalase activities and higher vitamin E and lower ubiquinone levels. At the concentration used, CUH did not significantly affect cell number or viability of melanocytes after either period of culture. On the contrary, vitiligo melanocytes were susceptible to the toxic effect of CUH after 24 h of continuous treatment at concentrations greater than 6.6 microM. The degree of CUH toxicity correlated strictly with the anti-oxidant pattern, defined as the ratio between vitamin E concentration and catalase activity, suggesting that the alteration in the antioxidants was the basis for sensitivity to the external oxidative stress. Our results demonstrate the presence of an imbalance in the anti-oxidant system in vitiligo melanocytes and provide further support for a free radical-mediated damage as an initial pathogenic event in melanocyte degeneration in vitiligo.

Antioxidant pattern in uveal melanocytes and melanoma cell cultures.

PURPOSE: To investigate the antioxidant status of cultured uveal melanocytes from patients with uveal melanoma and uveal melanoma cells to characterize some of the biochemical properties of these cells in respect to the normal cutaneous melanocytes. METHODS: The fatty acid pattern of membrane phospholipids, intracellular vitamin E level, and superoxide dismutase (SOD) and catalase activities were studied in uveal melanocytes (n = 10) and uveal melanoma cell (n = 10) cultures, by gas chromatography mass spectrometry or by spectrophotometer. RESULTS: Among the uveal melanocyte cultures, two groups were differentiated, according to catalase activity: group A with catalase values comparable to those of cutaneous ones and higher SOD activity and group B with catalase values 2 SD lower (P<0.001) and lower SOD activity. Vitamin E concentration was not significantly different between melanoma cells and melanocytes, whereas a significantly higher percentage of polyunsaturated fatty acids was found in melanoma cells and the B group of melanocytes (P = 0.022). In uveal melanoma cells SOD activity was significantly lower than that detected in uveal melanocytes (P< 0.005). CONCLUSIONS: These results show a different pattern of antioxidants in uveal melanocytes with respect to cutaneous ones, possibly related to the anatomic distribution. However, as in cutaneous melanocytes, two subgroups were identified on the basis of the antioxidant pattern that could be the expression of a constitutional increased susceptibility to oxidative stress in some subjects. Moreover, an imbalance of the antioxidants was observed in melanoma cells, possibly related to the disease status and progression.